Viral oncolysis, the destruction of cancer cells by replicating viruses, is under clinical investigation for cancer therapy. Lytic viral replication in cancer cells both destroys the cells and liberates progeny virion to infect adjacent cancer cells. The safety and efficacy of this approach are dependent on selective and robust viral replication in cancer cells rather than in normal cells. Methods to detect and quantify viral replication in tissues have relied on organ sampling for molecular analyses. Preclinical and clinical studies of viral oncolysis will benefit significantly from development of a noninvasive method to repetitively measure viral replication. We have shown that positron emission tomography (PET) allows for in vivo detection of herpes simplex virus (HSV)-1 replication in tumor cells using 9-(4-[(18)F]-fluoro-3-[hydroxymethyl]butyl)guanine ([(18)F]FHBG) as the substrate for HSV thymidine kinase (HSV-TK). As expected, phosphorylated [(18)F]FHBG is initially trapped within HSV-1-infected tumor cells and is detectable as early as 2 h following virus administration. MicroPET images reveal that [(18)F]FHBG accumulation in HSV-1-infected tumors peaks at 6 h. However, despite progressive accumulation of HSV-1 titers and HSV-TK protein in the tumor as viral oncolysis proceeds, tumor cell degradation resulting from viral oncolysis increases over time, which limits intracellular retention of [(18)F]FHBG. These observations have important consequences with regard to strategies to use [(18)F]FHBG PET for monitoring sites of HSV-TK expression during viral oncolysis.